Outbreak of Chagas disease in Brazil: Validation of a molecular diagnostic method.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), affects millions of people worldwide. Polymerase Chain Reaction (PCR) and real-time quantitative PCR (qPCR) have been used as tools to monitor parasitic levels in the bloodstream of individuals exposed to infection, thus enabling the monitoring of relapses and the effectiveness of therapy, for example. The aim of this study was to evaluate the TcSAT-IAM system, developed by our research group, on samples from patients with suspected Chagas disease infection. Initially, primer systems were developed for the detection of the nuclear DNA (SAT-DNA) from T. cruzi (TcSAT-IAM). The Cruzi system, predicted in the literature, and TcSAT-IAM were then evaluated in relation to their analytical sensitivity, specificity and efficiency. Afterwards, the applicability of the qPCR technique using both systems (separately) for the diagnosis of acute CD was evaluated in samples from 77 individuals exposed to the outbreak that occurred in Pernambuco-Brazil, relating the results obtained to those of the classical diagnostic methods recommended for this stage of the infection. TcSAT-IAM and Cruzi had a detection limit of 1 fg of target DNA (0,003 parasites). Thirty-eight cases were recorded, 28 by laboratory criteria and 10 by clinical and epidemiological criteria. Blood samples from 77 subjects were submitted to qPCR by both systems, reaching an agreement of 89.61% between them. After analyzes between systems and diagnostic criteria, the TcSAT-IAM showed sensitivity and specificity of 52.36% (CI 37.26-67.52) and 92.31% (CI 79.68-97.35), respectively, accuracy of 72.73% and moderate agreement. The TcSAT-IAM showed an accuracy of 72.58% and 75% in relation to parasitological and serological tests (IgM anti-T. cruzi), respectively. Therefore, quantitative PCR should be incorporated into the diagnosis of suspected acute cases of Chagas disease.

Clinical-therapeutic follow-up of patients with American cutaneous leishmaniasis caused by different Leishmania spp. in Brazil.

American cutaneous leishmaniasis (ACL) may present different clinical manifestations, immune and therapeutic responses, depending on the Leishmania species, as well as inoculum size and factors inherent to the affected individual. Thus, the aim of this study was to carry out clinical-therapeutic follow-up of Brazilian patients with ACL caused by different Leishmania species. Between 2015 and 2018, patients with ACL from Amazonas and Pernambuco states (Brazil) were submitted to blood collection before and after treatment. The qPCR technique was used to quantify the parasite load. To identify the Leishmania species, one of the following techniques was employed: a conventional PCR performed from biopsy or blood DNA, followed by sequencing; or Multilocus Enzyme Electrophoresis from Leishmania isolated from biopsy/aspirated lesion. A total of 10.8% (23/213) of the patients included in positive cases were followed-up. All 23 patients were clinically and epidemiologically compatible with ACL and were also positive in parasitological tests (86.96%), molecular tests (73.91%) or both (60.87%). Seventeen samples collected before treatment and 11 collected after treatment were positive in the qPCR assay, with a mean parasite load (MPL) of 38.33 fg/µL and 11.81 fg/µL, respectively. Eight samples were positive in both collections. Thirteen patients (56.52%) were clinically cured (wound healing). Ten patients (43.47%) were not clinically cured at the time of return with the attending physician. Identification of Leishmania species was carried out in samples from nine patients, and six were identified as L. (Viannia) braziliensis, 2 as L (Viannia) guyanensis and 1 as L (Leishmania) amazonensis. One patient infected with L. guyanensis and other with L. braziliensis were not clinically cured and increased the mean parasite load after treatment. The data obtained from the followed-up patients and the relationship between clinical evolution and the infecting species demonstrate the need to understand its etiology to define the effective therapeutic protocol.

Duplex qPCR for Leishmania species identification using lesion imprint on filter paper.

BACKGROUND: American cutaneous leishmaniasis (ACL) is caused by different Leishmania parasites, which stimulate and direct the immune response against the infection. OBJECTIVE: To evaluate the TaqMan probe technology applicability to diagnose and identifying of Leishmania spp. related to the ACL etiology. METHODOLOGY: Through the MEGA 6.0 software, performed an in silico analysis using multiple alignments of Leishmania spp. which were available on GenBank for different genomic targets. The efficiency (e), specificity and detection limit (DL) were calculated for each system, these were associated to compose a duplex-qPCR (DqPCR). The samples of blood, lesion biopsy and lesion imprint on filter paper from patients residing in states of Amazonas (AM) and Pernambuco (PE)-Brazil, (cases and controls) were used to perform the DqPCR technique. The capacity to identify the Leishmania species was determined by comparison with isoenzymes method and sequencing analysis. RESULTS: Internal Transcribed Spacer 1 (rDNA) was the target selected. Two sets of primers and probes were designed and combined: SVS for subgenus Viannia and LaS for L. (L.) amazonensis. The results were: SVSe = 93.24%, SVS DL = 50 fg/µL; LaSe = 89.3%, LaSLD = 5 fg/µL presented 100% of specificity. In total, 236 individuals participated of the present study, wherein were 101 blood samples, 33 biopsies and 147 lesion imprints. The imprint was the most sensitive sample, showing 83.06% of sensitivity, 86.96% of specificity and substantial agreement between the techniques analysis (k = 0.531; p < 0,001). Regarding the species identification, DqPCR and sequencing/isoenzymes have agreed at 100%, since the infection is caused by a single Leishmania species. CONCLUSION: The DqPCR technique was applicable in diagnosis and identification of Leishmania spp. (subgenus Viannia and L. amazonensis). Furthermore, the lesion imprint is less invasive, allowing a fewer discomfort and greater acceptance by the patients, in addition of being low cost and easy handling.

Chlorella vulgaris extract and Imiquimod as new therapeutic targets for leishmaniasis: An immunological approach.

The therapeutic regimen for the treatment of American Tegumentary Leishmaniasis (ATL) is targeted at the death of the parasite; therefore, it is essential to develop a treatment that can act on the parasite, combined with the modulation of the inflammatory profile. Thus, the aim of this study was to make an in vitro evaluation of the therapeutic potential of Chlorella vulgaris extract (CV) and Imiquimod for ATL. Selectivity indices (SI) were determined by inhibitory concentration assays (IC50) in L. braziliensis cells and cytotoxic concentrations (CC50) were measured in human cells using the MTT method, based on the CV microalgae extract (IC50 concentrations of 15.63 to 500 µg/mL; CC50 concentrations of 62.5-1000 µg/mL) in comparison with the reference drugs and Imiquimod. The immune response was evaluated in healthy human cells by gene expression (RT-qPCR) and cytokine production (Flow Cytometry). The CV extract (SI = 6.89) indicated promising results by showing higher SI than meglumine antimoniate (SI = 3.44) (reference drug). In all analyses, CV presented a protective profile by stimulating the production of Th1 profile cytokines to a larger extent than the reference drugs. Imiquimod showed a high expression for Tbx21, GATA3, RORc and Foxp3 genes, with increased production only of the TNF cytokine. Therefore, the data highlight the natural extract and Imiquimod as strong therapeutic or adjuvant candidates against ATL, owing to modulation of immune response profiles, low toxicity in human cells and toxic action on the parasite.

Detection of Leishmania infantum amastigotes in neutrophil from peripheral blood in a naturally infected dog.

Canine visceral leishmaniasis (CVL) is a zoonotic disease of high lethality caused by Leishmania infantum in the Americas. In the infected dog, the amastigotes are scarce in blood, especially in the late phase of the disease. This study aimed to report a rare case of L. infantum amastigotes found in neutrophils from peripheral blood of a naturally infected dog in terminal phase of CVL, also describing its clinical status before and after treatment with miltefosine 2%. The dog, which presented as polysymptomatic and with classical signs and symptoms of CVL was submitted to the following tests: Dual Path Platform (DPP) rapid test, ELISA and parasitological examination of peripheral blood. Hematological and biochemical parameters were obtained before and after treatment. All diagnostic tests were positive for CVL. The identification of L. infantum amastigotes inside neutrophils from peripheral blood was confirmed through microscopy, and the species was confirmed by molecular analysis. At the end of the treatment, peripheral parasitemia was not detected, and improvements were observed in clinical and laboratorial parameters. Finally, this atypical finding can be used as example to raise discussions about the real immunological role of neutrophils in late phases of CVL and its clinical/therapeutic implications.

Clinical and epidemiological profiles of patients with American cutaneous leishmaniasis from the states of Pernambuco and Amazonas, Brazil.

INTRODUCTION: Brazil has a high number of cases of American cutaneous leishmaniasis (ACL) in the north and northeast regions. Therefore, continuous surveillance of environmental and socioeconomic factors in endemic areas is needed to develop strategic control measures. This study aimed to describe the clinical and epidemiological profiles of patients with ACL. METHODS: All patients were from the states of Amazonas and Pernambuco, and examinations were carried out between 2015 and 2018. All patients had a clinical and epidemiological history compatible with ACL after positive diagnostic tests. Information obtained from medical records included gender, employment activity, level of education, age, and number and sites of lesions. RESULTS: A total of 213 patients were included, of whom 30.98% were female and 69.02% were male. The main employment activity was agriculture (27.56%). The most common level of education was elementary (62.42%). The average age was approximately 39 years. The majority of the patients presented only with one lesion (54.87%), and legs/feet were the most commonly affected area (48.25%), followed by the arms/hands (44.75%). CONCLUSIONS: These data demonstrated that irrespective of the patients' places of origin, interventions need to be focused on men of economically productive age, in view of the high risk of exposure to the vector in this group. Education activities need to be directed to farmers about the importance of protection against ACL vectors during work. Such information must also be directed to employers as a way of implementing and maintaining appropriate working conditions and stepping up vector control.

Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach.

INTRODUCTION:: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS:: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS:: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS:: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.

Clinical and epidemiological profiles of patients with American cutaneous leishmaniasis from the states of Pernambuco and Amazonas, Brazil

Abstract INTRODUCTION: Brazil has a high number of cases of American cutaneous leishmaniasis (ACL) in the north and northeast regions. Therefore, continuous surveillance of environmental and socioeconomic factors in endemic areas is needed to develop strategic control measures. This study aimed to describe the clinical and epidemiological profiles of patients with ACL. METHODS: All patients were from the states of Amazonas and Pernambuco, and examinations were carried out between 2015 and 2018. All patients had a clinical and epidemiological history compatible with ACL after positive diagnostic tests. Information obtained from medical records included gender, employment activity, level of education, age, and number and sites of lesions. RESULTS: A total of 213 patients were included, of whom 30.98% were female and 69.02% were male. The main employment activity was agriculture (27.56%). The most common level of education was elementary (62.42%). The average age was approximately 39 years. The majority of the patients presented only with one lesion (54.87%), and legs/feet were the most commonly affected area (48.25%), followed by the arms/hands (44.75%). CONCLUSIONS: These data demonstrated that irrespective of the patients' places of origin, interventions need to be focused on men of economically productive age, in view of the high risk of exposure to the vector in this group. Education activities need to be directed to farmers about the importance of protection against ACL vectors during work. Such information must also be directed to employers as a way of implementing and maintaining appropriate working conditions and stepping up vector control.

Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach

Abstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.

Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis

Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.

Aplicabilidade da técnica de PCR em tempo real para caracterização de espécies de Leishmania / Aplicability of real time PCR technique for leishmania species characterization

A Leishmaniose Tegumentar Americana (LTA) é causada por protozoários, do gênero Leishmania, envolvidos em um complexo ciclo biológico. No Brasil, sete espécies estão envolvidas com a etiologia da doença, distribuídas em todas as regiões geográficas e responsáveis por diferentes manifestações clínicas. Diante disso, o diagnóstico em conjunto com a identificação da espécie é de grande importância clínico-terapêutica. Este trabalho tem por objetivo avaliar a aplicabilidade da técnica de PCR quantitativa em tempo real (qPCR) para identificação de espécies de Leishmania envolvidas com a etiologia da LTA. Foram realizados ensaios de qPCR para padronização da Temperatura de melting (Tm) utilizando cepas de referência de diferentes espécies de Leishmania. Após o diagnóstico em amostras de sangue de animais domésticos utilizando a qPCR, as amostras positivas foram analisadas através de suas Tm, e os produtos de qPCR foram purificados e sequenciados. Dez amostras previamente caracterizadas por isoenzimas, também foram analisadas através da Tm. Ainda como teste de referência, foi padronizada uma Restriction Fragment Length Polymorphism (RFLP) utilizando as cepas de referência e testada nas amostras. Através da padronização da Tm das espécies, foram criados dois intervalos de análise: 1 (Tm = 78-79,99°C), que compreende: Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis e Leishmania (V.) shawi; e 2 (Tm = 80-82,2°C), que compreende: Leishmania (V.) naiffi, Leishmania (L.) amazonensis e Leishmania (L.) mexicana. Um total de 223 amostras positivas foi analisado, destas, 58 incluídas no intervalo 1 e 165 no intervalo 2. O sequencimento de 94 destas amostras foi correspondente à L. (V.) braziliensis, L. (V.) panamensis e L. (V.) guyanensis. A RFLP em 173 amostras identificou 167 L. (V.) braziliensis, 05 L. (L.) mexicana e 01 L. (V.) panamensis...

The American Cutaneous Leishmaniasis (ACL) is caused by protozoa of the genus Leishmania, involved in a complex biological cycle. In Brazil, seven species are involved in the etiology of the disease, distributed in all geographic regions and responsible for different clinical manifestations. Therefore, the diagnosis together with the identification of the species is of great clinical and therapeutic importance. This work aims to evaluate the applicability of the technique of real-time quantitative PCR (qPCR) for the identification of Leishmania species involved in the etiology of ACL. qPCR assays for standardizing the melting temperature (Tm) using reference strains of different species of Leishmania were performed. After the diagnosis on blood samples of domestic animals using the qPCR positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten samples previously characterized by isoenzymes were also analyzed by Tm. Also as a reference test was standardized as Restriction Fragment Length Polymorphism (RFLP) using the reference strains and tested on samples. Through standardization of Tm species two ranges of analysis were created: 1 (Tm = 78-79,99°C), comprising: Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis e Leishmania (V.) shawi; and, 2 (Tm = 80-82,2°C), comprising: Leishmania (V.) naiffi, Leishmania (L.) amazonensis e Leishmania (L.) mexicana. A total of 223 positive samples were analyzed, of these, 58 included in the range 1 and 165 in the range 2 to 94 and the sequence of these samples corresponded to L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis. By RFLP in 173 samples were identified 167 L. (V.) braziliensis, 05 L. (L.) mexicana and 01 L. (V.) panamensis The analysis of Tm of the ten samples characterized by isoenzymes showed 80% agreement (p = 0.6499) between the gold standard (isoenzymes) and intervals developed in this study...